DESCRIPTION: This project proposes the development of a microfluidic ELISA method that will allow early diagnosis of Alzheimer's disease (AD) through highly sensitive and specific detection of amyloid--derived diffusible ligands (ADDL) in human cerebrospinal fluid (CSF). The proposed enhancement in ADDL detection will be accomplished based on a three-fold strategy that will focus on 1) improving the specific capture of ADDL molecules, 2) reducing the non-specific binding of the detection antibody to the assay surface and 3) depleting the amount of amyloid- (A) monomer in the sample prior to analysis to diminish its cross- reactivity with the capture/detection antibody. Interestingly, all of these goals will be accomplished in our project based on an electric field assisted immunocapture process that has been recently demonstrated in the PI's laboratory. This approach relies on the electrokinetic focusing of a target protein around a semi-permeable membrane to significantly increase its local concentration over an ELISA surface created next to the membrane structure, improving the immunocapture of the protein species by over two orders of magnitude. Our preliminary work shows that upon application of this strategy to a standard sample obtained as part of a commercial ELISA kit, the limit of detection for A oligomers can be reduced by a factor of about 122. Building on this result, we propose the application of the above- described pre-concentration process to improving the immunocapture of ADDL molecules on the ELISA surface as well as depleting the amount of A monomers from human CSF samples to allow reliable detection of AD. In addition, we will also develop a novel approach to reducing the non-specific binding of the detection antibody in our assays by decreasing its incubation period. Because non-specific interactions among proteins tend to be much weaker than the specific ones between an antigen-antibody pair, a decrease in this incubation time is expected to reduce the non-specific adsorption of the detection antibody on our ELISA surface significantly more than its specific capture by the analyte molecule. Moreover, any reduction in signal due to the latter effect will be compensated in our approach through electrokinetic pre-concentration of the enzyme reaction product around a membrane interface. The performance of the proposed ELISA method will be assessed by applying it to quantitatively determining ADDL levels in human CSF samples obtained through our collaborator, Dr. Christopher Filley, from the Denver VA Medical Center. Fortuitously, our collaborator has access to several CSF samples from human subjects who did not exhibit any symptoms of AD at the time of sample collection but later developed the disease. The reason for CSF sample collection from these subjects was the fact that they were genetically prone to developing AD. The ability of our microfluidic device to correlate the ADDL level in these samples to the progression of the disease in the corresponding human subjects will establish its utility as a diagnostic tool for early detection of AD.